Shigella species and enteroinvasive E. coli invade and multiply within intestinal epithelial cells of their human host. The bacteria are able to spread directly to adjacent epithelial cells and the invasion and resulting host inflammatory response cause the symptoms of dysentery, a disease responsible for significant morbidity and mortality worldwide. These invasive pathogens are able to rapidly adapt to the intracellular environment of the host and acquire the essential nutrients for their survival. In particular, they must compete with the host for the essential element iron. Shigella and pathogenic E. coli have multiple mechanisms for iron acquisition. These include the synthesis and transport of low molecular weight iron binding compounds called siderophores and transport of ferric iron, ferrous iron or heme. One of the goals of this project is to define and more fully characterize these systems for iron acquisition. In addition, we will determine the mechanism of regulation of expression of these systems and determine how iron regulates a large number of genes within the bacterial cells. We have shown that an iron-binding represser protein (Fur) and a small RNA (RyhB) are involved in regulation of gene expression, including regulation of virulence genes. Microarray analysis and genetic screens will be used to reveal the full spectrum of genes regulated by iron, Fur, and RyhB in these pathogens. We have found that RyhB regulates the expression of a number of genes, including virulence plasmid genes that encode invasion proteins and the type III secretion apparatus, and chromosomal genes involved in acid resistance. These affects appear to be the result of RyhB repression of the virulence gene transcription factor VirB and the acid resistance regulator EvgAS. We will determine the mechanism by which RyhB controls the expression of virB and evgAS. These studies will help define the global regulatory patterns that allow Shigella to respond to the various environments that it encounters in the host such as the acidity of the stomach and the low iron environment within host cells.